1. Field of the Invention
The present invention relates generally to the fields of genetic engineering and protein secretion. More specifically, the present invention relates to engineering of leader peptides for the secretion of recombinant proteins in bacteria.
2. Description of the Related Art
Proteins destined for secretion from the cytoplasm are synthesized with an N-terminal peptide extension of generally between 15–30 amino acids known as the leader peptide. The leader peptide is proteolytically removed from the mature protein either concomitant to or immediately following export into an exocytoplasmic location.
Recent findings have established that there are actually four protein export pathways in Gram-negative bacteria (Stuart and Neupert, 2000): the general secretory (Sec) pathway (Danese and Silhavy, 1998; Pugsley, 1993), the signal recognition particle (SRP)-dependent pathway (Meyer et al., 1982), the recently discovered YidC-dependent pathway (Samuelson et al., 2000) and the twin-arginine translocation (Tat) system (Berks, 1996). With the first three of these pathways, polypeptides cross the membrane via a ‘threading’ mechanism, i.e., the unfolded polypeptides insert into a pore-like structure formed by the proteins SecY, SecE and SecG and are pulled across the membrane via a process that requires the hydrolysis of ATP (Schatz and Dobberstein, 1996).
In contrast, proteins exported through the Tat-pathway transverse the membrane in a partially or perhaps even fully folded conformation. The bacterial Tat system is closely related to the ‘ΔpH-dependent’ protein import pathway of the plant chloroplast thylakoid membrane (Settles et al., 1997). Export through the Tat pathway does not require ATP hydrolysis and does not involve passage through the SecY/E/G pore. In most instances, the natural substrates for this pathway are proteins that have to fold in the cytoplasm in order to acquire a range of cofactors such as FeS centers or molybdopterin. However, proteins that do not contain cofactors but fold too rapidly or too tightly to be exported via any other pathway can be secreted from the cytoplasm by fusing them to a Tat-specific leader peptide (Berks, 1996; Berks et al., 2000).
The membrane proteins TatA, TatB and TatC are essential components of the Tat translocase in E. coli Sargent et al., 1998; Weiner et al., 1998). In addition, the TatA homologue TatE, although not essential, may also has a role in translocation and the involvement of other factors cannot be ruled out. TatA, TatB and TatE are all integral membrane proteins predicted to span the inner membrane once with their C-terminal domain facing the cytoplasm. The TatA and B proteins are predicted to be single-span proteins, whereas the TatC protein has six transmembrane segments and has been proposed to function as the translocation channel and receptor for preproteins (Berks et al., 2000; Bogsch et al., 1998; Chanal et al., 1998). Mutagenesis of either TatB or C completely abolishes export (Bogsch et al., 1998; Sargent et al., 1998; Weiner et al., 1998). The Tat complex purified from solubilized E. coli membranes contained only TatABC (Bolhuis et al., 2001). In vitro reconstitution of the translocation complex demonstrated a minimal requirement for TatABC and an intact membrane potential (Yahr and Wickner, 2001).
The choice of the leader peptides, and thus the pathway employed in the export of a particular protein, can determine whether correctly folded functional protein will be produced (Bowden and Georgiou, 1990; Thomas et al., 2001). Feilmeier et al. (2000) have shown that fusion of the green fluorescent protein (GFP) to a Sec-specific leader peptide or to the C-terminal of the maltose binding protein (MBP which is also exported via the Sec pathway) resulted in export of green fluorescent protein and MBP-GFP into the periplasm. However, green fluorescent protein in the periplasm was non-fluorescent indicating that the secreted protein was misfolded and thus the chromophore of the green fluorescent protein could not be formed. Since proteins exported via the Sec pathway transverse the membrane in an unfolded form, it was concluded that the environment in the bacterial secretory compartment (the periplasmic space) does not favor the folding of green fluorescent protein Feilmeier et al., 2000). In contrast, fusion of a Tat-specific leader peptide to green fluorescent protein resulted in accumulation of fluorescent green fluorescent protein in the periplasmic space. In this case, the Tat-GFP propeptide was first able to fold in the cytoplasm and then be exported into the periplasmic space as a completely folded protein (Santini et al., 2001; Thomas et al., 2001). However, there has been no evidence that leader peptides other than TorA can be employed to export heterologous proteins into the periplasmic space of E. coli. 
The cellular compartment where protein folding takes place can have a dramatic effect on the yield of biological active protein. The bacterial cytoplasm contains a large number of protein folding accessory factors, such as chaperones whose function and ability to facilitate folding of newly synthesized polypeptides is controlled by ATP hydrolysis. In contrast, the bacterial periplasm contains relatively few chaperones and there is no evidence that ATP is present in that compartment. Thus many proteins are unable to fold in the periplasm and can reach their native state only within the cytoplasmic milieu. The only known way to enable the secretion of folded proteins from the cytoplasm is via fusion to a Tat-specific leader peptide. However, the protein flux through the Tat export system is significantly lower than that of the more widely used Sec pathway. Consequently, the accumulation and steady state yield of proteins exported via the Tat pathway is low.
The prior art is thus deficient in methods of directing efficient export of folded proteins from the cytoplasm. The present invention fulfills this long-standing need and desire in the art.